1. Field of the Invention
The present invention relates to a method for testing microorganisms or the like and a testing apparatus therefor, and particularly relates to a microorganism-testing apparatus using a fluorescent cytometry method. For the purpose of the present specification, the microorganism-testing method and the testing apparatus therefor refer to a method for testing cells or microorganisms and an testing apparatus therefor and are not intended to be limited to a method for testing only microorganisms and an testing apparatus therefor. For simplicity of indication, the “cells or microorganisms” may be simply indicated with “microorganisms” in the present specification and what is claimed is, in which case the “microorganisms” shall include the “cells”.
2. Background Art
Viable bacteria-counting apparatuses (microorganism-testing apparatuses) are provided on the market which are intended for the speed-up and simplification of counting viable bacteria in a food. Among these, attention has been given to viable bacteria-counting apparatuses using fluorescent cytometry method as viable bacteria-counting apparatuses excellent in rapidity and quantitativity. Fluorescent cytometry methods are methods involving counting fluorescently-stained viable bacteria on a one-by-one basis in a short time and roughly classified into a fluorescent image cytometry method and a fluorescent flow cytometry method. The fluorescent image cytometry method is a method which involves adsorbing fluorochrome-stained viable bacteria onto a glass, filter, or the like and counting the adsorbed viable bacteria on a one-by-one basis. On the other hand, the fluorescent flow cytometry method is a method which involves making the flow diameter of a sample fluid containing fluorochrome-stained viable bacteria thin to count the viable bacteria flowing through a passage on a one-by-one basis.
A problem in measuring the number of viable bacteria in a food with the viable bacteria-counting apparatus using fluorescent cytometry is the requirement of purification for removing contaminants before counting. Contaminants include killed bacteria and particles of a fluorochrome used for staining in addition to materials derived from the food such as chloroplasts, chromatophores, mitochondria, carbohydrates (starch and glycogen), and a lipid mass. These contaminants contribute to the miscounting thereof as viable bacteria because they emit autofluorescence or fluorescence by being stained by, or adsorbing to, a fluorochrome. Methods for purifying viable bacteria include a centrifugation method and a column method, and these methods can separate viable bacteria and contaminants with a high purification degree.
In contrast, as an example of discriminating between viable bacteria and contaminants without performing purification, there is a method which involves discriminating between viable bacteria and killed bacteria using two types of fluorochromes to count viable bacteria. This method is a discrimination method based on the fact that although damage is present on the membrane surface of killed bacteria, damage is absent on the membrane surface of viable bacteria, and involves discriminating between viable bacteria and killed bacteria using a membrane-permeable blue fluorochrome, DAPI (4′, 6-diamidine-2′-phenylindole), and a membrane-impermeable red fluorochrome, PI (propidium iodide). The blue fluorescence of DAPI is emitted from the viable bacteria and the killed bacteria because the membrane-permeable DAPI permeates the membrane of bacteria with or without damage, while the red fluorescence of PI is emitted only from the killed bacteria because the membrane-impermeable PI permeates only the damaged membrane thereof. A method involving specifically staining materials with a plurality of fluorochromes having different wavelengths to discriminate between the materials based on the difference in the wavelength between their fluorescences as in the above method is called a multi-staining method.
JP Patent Publication (Kokai) Nos. 2008-157829 A and 2009-178078 A disclose fluorescent flow cytometry methods using a technique involving holding a sample and a reagent(s) in a microorganism-testing chip to simply test microorganisms.